Peripheral blood is the most frequently accessed tissue in the clinic, and the isolation of blood-borne cells is of broad clinical and scientific importance in hematology, transfusion, immunology, regenerative medicine, and oncology. Recent developments in microengineering have greatly advanced our capabilities in isolating pure populations of cells and performing high-throughput, multidimensional assays (1). The rapidly growing field of blood-on-a-chip technologies has expanded into applications ranging from T cell isolation for HIV disease monitoring (2) and multiplexed detection of cytokine secretion (3); gene expression profiling of neutrophils in trauma and burn patients (4); enrichment of CD34+ hematopoietic stem cells (5); to minimally invasive detection of nucleated red blood cells (RBCs) from maternal blood (6) as well as rare circulating tumor cells for cancer diagnosis (7) and identification of druggable mutations (8).
Similar to any tissue, however, whole blood (WB) deteriorates quickly ex vivo. Degradation events such as nutrient deprivation, oxidative stress, changes in osmolarity and pH, and accumulation of toxic metabolic byproducts quickly commence. Within hours, neutrophils undergo activation, oxidative bursting, as well as necrosis and apoptosis. Blood settling causes physical stress, and exacerbate degradation by mechanically compacting necrotic cells in a confined space that accelerates collateral damage and cross-activation. Transportation of blood samples results in uncontrolled shaking which induces hemolysis and platelet activation. These damages not only affect the viability and functionality of the cells of interest in the sample, but also immensely impact the enrichment technologies in a wide spectrum of applications. For instance, shedding of surface antigens render antibody-based sorting ineffective. Red blood cell rouleaux formation may trap rare cells. In particular, microfluidic sorting technologies which are essential for efficient cell sorting are compromised by echinocytes (aged red blood cells that form spiculations), platelet activation and clotting, as well as cellular aggregation.
To further complicate preservation strategies for whole blood, hypothermic temperature ranges which can otherwise effectively suppress biochemical reactions and degradation processes have been considered incompatible, due mainly to cold-induced platelet activation. As such, in modern transfusion medicine whole blood is typically stored in room temperature and processed into various components for specialized storage within 24 hours (9). Given that many clinically relevant assays such as sequencing and expression profiling are best performed in large medical centers or diagnostic laboratories, the logistical needs of blood storage and transportation impose severe limitations to the dissemination of next-generation blood-based medical technologies.
Among the many applications that will benefit tremendously from improved preservation of whole blood is the isolation of circulating tumor cells (CTCs), which are shed from solid tumors and capable of hematologic spread of metastasis. Advanced microfluidic technologies have enabled isolation of these extremely rare cells (one in a billion blood cells) from peripheral blood samples of cancer patients, and significant progress has been made in using these cells and their molecular signature for diagnosis, prognosis, identification of druggable mutations, as well as generation of patient-specific models for drug screening. However, these downstream assays are critically dependent upon the isolation of viable, unfixed CTCs that retain the molecular information and cellular function. Degradation of blood not only restricts molecular analysis of CTCs, but also interferes with the precise microfluidic isolation of these extremely rare cells.